Oligonucleotides functionalized 3′, 5′ or internally with reactive moieties have been used for a variety of therapeutic and diagnostic purposes. For example, modified oligonucleotides incorporating targeting moieties have been prepared to increase activity of antisense oligonucleotides for therapy. Functionalized or modified oligonucleotides have been prepared to permit conjugation to supports and also for preparation of conjugates.
Methods have been described for achieving these purposes, but in many cases these methods have inherent disadvantages and do not satisfy all the criteria required by currently developing technologies. Methods in which a functional moiety can be incorporated into an oligonucleotide during its synthesis on a solid support are limited. There is a need to develop methods to site-specifically bind oligonucleotides to surfaces for a variety of purposes, such as for identification of genes for pharmacogenomic, diagnostic and therapeutic purposes.
Functional groups that can be added directly to a growing oligonucleotide on a solid support include amines and thiols. Direct binding of amino or thiol-functionalized oligonucleotides to solid surfaces require that the surface be modified with a moiety that reacts directly with an amino or thiol function. The reactive moieties succinimidyl esters and maleimides have been used for this purpose. The coupling efficiency of these moieties suffers from their inherent instability and ease of hydrolysis (for maleimides and succinimidyl esters).
Biotin monomers that can be added to the 5′-end of an oligonucleotide on a solid support have been described, as well as coupling of biotinylated oligonucleotides to surfaces and other molecules that have been modified to incorporate either avidin or streptavidin protein. This method is functional but requires the use of a protein that is expensive, may lead to non-specific binding, and has at most four biotin binding sites per molecule. Also, incorporation of large molecules on biotin reduces the binding affinity and the number of accessible binding sites on avidin. The density of oligonucleotide per mm2 is limited due to the use of a protein having a molecular weight of about 14000.
These same limitations exist for the preparation of oligonucleotide conjugates. The incorporation of activated functionalities, such as succinimidyl esters or maleimides, of the conjugation partner (compound to be conjugated to the oligonucleotide) can be costly, and not even possible in some instances.
Table 1 below lists the available reactive couples to bind oligonucleotides to solid surfaces.
TABLE 15′-oligonucleotidemodificationSurface modificationaminoN-hydroxysuccinimidyl ester (NHS),isothiocyanates (ITC)thiolMaleimide, bromoacetyl, activated thiol(to form disulfide)disulfidethiolbiotinavidin
Due to the limitations of available methods, there is a need for efficient crosslinking agents for immobilization of oligonucleotides on solid surfaces, and for preparation of biopolymer conjugates.
Therefore, it is an object herein to provide reagents and methods for immobilization of oligonucleotides on solid surfaces without the need for post-synthetic modification of the oligonucleotide. It is also an object herein to provide reagents and methods for synthesis of biopolymer conjugates, including oligonucleotide, protein, oligosaccharide, glycoprotein and/or protein nucleic acid (PNA) conjugates. A further object herein is to provide modified, immobilized or conjugated oligonucleotides.